The present invention relates generally to flow cytometry. More specifically, it relates to the simultaneous measurement of incorporated nucleoside analogs and the total cellular deoxyribonucleic acid (DNA) content by flow cytometric techniques.
A broad range of biological and biomedical investigations depends on the ability to distinguish cells that synthesize DNA from those that do not. Oncologists, for example, have devoted substantial effort to establishing correlations between the proportion of human tumor cells synthesizing DNA and treatment prognosis, e.g. Hart et al., Cancer, Vol. 39, pgs. 1603-1617 (1977). Effort has also been devoted to improvement of anticancer therapy with S-phase specific agents by treating when the experimentally determined proportion of tumor cells in S phase is maximal, e.g. Barranco et al., Cancer Research, Vol. 42, pgs. 2894-2898 (1982). In these studies, S-phase cells are usually assumed to be those that appear labeled in autoradiographs prepared immediately after pulse labeling with tritiated thymidine, or those with S-phase DNA content in DNA distributions measured flow cytometrically. Cancer researchers and oncologists have relied heavily on measurements of the proportion of DNA synthesizing cells to determine the cell cycle traverse characteristics of normal and malignant cells. The classical "fraction of labeled mitosis" procedure, Quastler et al., Experimental Cell Research, Vol. 17, pgs. 420-429 (1959), for example, depends on assessment of the frequency of mitotic cells that appear radioactively labeled in autoradiographs of samples taken periodically after labeling with tritiated thymidine. Studies of the cell cycle traverse characteristics of drug-treated cell populations typically require measurement of the amount of tritiated thymidine incorporated by cells in S phase (e.g., by liquid scintillation spectrometry) or determination of the fraction of cells with S-phase DNA content (e.g., by DNA distribution analysis), or both, Pallavicini et al., Cancer Research, Vol. 42, pgs. 3125-3131 (1982). Studies of mutagen-induced genetic damage that use unscheduled DNA synthesis as an index of damage also rely on the detection of low levels of incorporation of tritiated thymidine, e.g. Painter et al., Biochim. Biophys. Acta, vol. 418, pgs. 146-153 (1976).
These broad-ranging biomedical studies are often limited by the measurement techniques. For example, autoradiographic determination of the fraction of cells incorporating radioactive DNA precursors like tritiated thymidine is limited by the labor-intensive nature of the measurements and by the subjectivity associated with discrimination between unlabeled and weakly labeled cells, e.g. Simpson-Herren, et al., Cancer Research, Vol. 36, pgs. 4705-4709 (1976). Determination of the amount of radioactive DNA precursor incorporation by liquid scintillation is also suboptimal because no information is available about the distribution of radioactivity incorporation among the cells of the population. Finally, interpretation of DNA distributions measured by flow cytometry is difficult because no information is available about the absolute rate of DNA synthesis, Gray et al., in Flow Cytometry and Sorting, Melamed et al., Eds. (Wiley, New York, 1979) pgs. 383-407. Thus, a population proliferating with twice the rate of another might have the same DNA distribution if the two populations spend the same fractional times in the G.sub.1, S, and G.sub.2 M phases of the cell cycle. Furthermore, discrimination between actively synthesizing and quiescent cells with S-phase DNA content is impossible.
Recently several fluorometric procedures have been developed to overcome some of the limitations associated with techniques for detecting DNA synthesis that depend on incorporated radionucleotides. Darzynkiewicz and co-workers have attempted to distinguish non-cycling cells from cycling cells by the degree of denaturation of cellular DNA after treatment with acid. The degree of denaturation is measured by the dye acridine orange which emits green fluorescence upon intercalation into double stranded sections of the DNA, and which emits red fluorescence upon electrostatic binding to single stranded sections of DNA. Total cellular DNA is correlated to the sum of the intensities of red fluorescence and green fluorescence, Darzynkiewicz et al., "Thermal Denaturation of DNA In Situ as Studied by Acridine Orange Staining and Automated Cytofluorometry," Experimental Cell Research, Vol. 90, pgs. 411-428 (1975); and Darzynkiewicz et al., "Different Sensitivity of Chromatin to Acid Denaturation in Quiescent and Cycling Cells as Revealed by Flow Cytometry," J. Histochem, Cytochem., Vol. 27, pgs. 478-485 (1979).
Latt et al., in "Flow cytometric analysis of bromodeoxyuridine - substituted cells stained with 33258 Hoechst," J. Histochem Cytochem., Vol. 25, pg. 927 (1977), and later Noguchi et al., in "Measurement of DNA Synthesis by Flow Cytometry," Cytometry, Vol. 1, pgs. 390-393 (1981), have shown that the incorporation of bromodeoxyuridine (BrdU) can be detected flow cytometrically by its quenching effect on the fluorescence from the DNA-specific dye Hoechst 33258. This technique has been used to quantify the rates of cell cycle traverse by measuring Hoechst 33258 fluorescence distributions for cells grown continuously in medium containing BrdU, e.g. Bohmer, "Flow Cytometric Cell Cycle Analysis Using the Quenching of 33758 Hoechst Fluorescence by Bromodeoxyuridine Incorporation," Cell and Tissue Kinetics. Vol. 12, pgs. 101-110 (1979); and Rabinovitch,"Regulation of Human Fibroblast Growth Rate by Both Noncycling Cell Fraction and Transition Probability Is Shown by Growth in 5-Bromodeoxyuridine Followed by Hoechst 33258 Flow Cytometry," Proc. Natl. Acad. Sci. Vol. 80, pgs. 2951-2955 (1983). Phase durations were estimated from the rate at which cells with Gl-, S-, and G2M-phase DNA contents moved through S-phase, incorporated BrdU and showed reduced Hoechst 33258 fluorescence. Unfortunately, these studies required incorporation of substantial amounts of BrdU and would be difficult to conduct in vivo because of BrdU toxicity.
The utility of BrdU as a marker for proliferating cells has been substantially increased by the development of antibodies against BrdU incorporated into cellular DNA, botn polyclonal: Gratzner et al., "The Use of Antibody Specific for Bromodeoxyuridine for the Immunofluorescent Determination of DNA Replication in Single-Cells and Chromosomes, " Experimental Cell Research, Vol. 95, pgs. 88-94 (1975); and monoclonal: Gratzner, "Monoclonal Antibody to 5-Bromo-and 5-Iododeoxyuridine: A New Reagent for Detection of DNA Replication," Science, Vol. 218, pgs. 474-475 (1982); Gratzner, U.S. Pat. No. 4,529,700 issued 16 July 1985; Raza et al., "Rapid Enumeration of S-Phase Cells by Means of Monoclonal Antibodies, " New England J. Medicine, Vol. 310, pg. 991 (1984); and Vanderlaan et al., copending U.S. patent application Ser. No. 542,967 filed 18 Oct. 1983. when coupled with fluorescent labels the antibodies are highly sensitive reagents for measuring the amount of BrdU incorporated into cellular DNA. Use of the antibodies requires that native double stranded DNA be altered so that the bromodeoxyuridine moieties of the nucleic acid are made accessible to the antibodies. Such alteration is usually accomplished by disrupting the hydrogen bonds between the two strands of the native DNA by standard denaturing techniques, e.g. see Gratzner et al., Experimental Cell Research, Vol. 95, pgs. 88-94 (1975) in regard to a denaturation protocol used for appliction of anti-BrdU antibodies; and Henderson, International Review of Cytology, Vol. 76, pgs. 1-46, for an extensive review of denaturation protocols used in in situ hybridization studies. Unfortunately, use of the antibodies alone does not provide a means for correlating BrdU incorporation with the phases of the cell cycle, a measurement which would be highly useful in distinguishing cycling from non-cycling cells, in studies for quantifying cell cycle parameters, and also in the analysis of populations perturbed by anti-cancer agents.